Nipah and Hednra viruses are lethal paramyxoviruses classified as Priority Pathogens and Emerging Infectious Disease agents. We propose a multidisciplinary project that capitalizes on a platform optimized by the PI (Dr. Benhur Lee) to study Nipah and Hendra virus entry at less than BSL4 levels. The project synergizes the molecular virology expertise of the PI with the unique resources and expertise of the co-Pis (Dr. Nathan Wolfe and Dr. Z. Hong Zhou) to shed light on a potentially broad spectrum of zoonotic viral pathogens. We will first focus on the henipaviruses as proof of principle for our approach, although we expect to be able to expand our analyses to other zoonoses like filioviruses, arenaviruses, hantaviruses and SARS. All three investigators are at UCLA. The objective of our proposal is to use our optimized Vesicular Stomatitis Virus (VSV) reporter platform in two complementary sets of studies: (1) for structural virology studies that enhance our understanding of the henipaviral fusion cascade, and (2) for higher throughput assays to detect neutralizing and/or cross-reactive antibodies for diagnostics, surveillance, and vaccine development efforts. The underlying rationale for our proposal is that serum neutralization assays and structural virology studies done with our VSV-rluc (Renilla luciferase) reporter pseudotypes will provide biologically relevant information more efficiently and economically than using their live viral counterparts. To accomplish our stated objectives, we propose the following Specific Aims: (1) To gain a supramolecular nanoscale understanding of the henipavirus fusion cascade using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) to visualize Nipah virus F and G oligomers pseudotyped onto VSV, and (2) To study relevant serum samples from the Global Viral Forecasting Initiative using our henipavirus VSV pseudotypes. Additionally, we have recently developed a catalytically improved beta-lactamase-Nipah matrix based assay for sensitive, specific and higher throughput analysis of native henipavirus entry and budding at BSL2 conditions. Thus, we also propose a Specific Aim (3): To identify and characterize small molecule inhibitors of henipavirus budding using our Nipah matrix VLP based assay. Our screen will be performed concurrently with Dr. Juan Carlos de la Torres' (another PI in our viral zoonoses thematic group) efforts to screen for small molecule inhibitors of arenavirus budding and any lead candidates can be cross-tested in each other's budding assay in order to identify putative broad spectrum inhibitors of viral budding.